RESEARCH ARTICLE
Yeast and yeast-like fungi associated with dry indehiscent fruits
of Nothofagus nervosa in Patagonia, Argentina
Natalia V. Fernández1,2, M. Cecilia Mestre1,2, Paula Marchelli2,3 & Sonia B. Fontenla1
Laboratorio de Microbiologı́a Aplicada y Biotecnologı́a, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue – INIBIOMA,
Rı́o Negro, Argentina; 2Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET), Capital Federal, Buenos Aires, Argentina; and
3
Unidad de Genética Ecológica y Mejoramiento Forestal, Instituto Nacional de Tecnologı́a Agropecuaria (INTA), Rı́o Negro, Argentina
1
Correspondence: Natalia V. Fernández,
Laboratorio de Microbiologı́a Aplicada y
Biotecnologı́a, Centro Regional Universitario
Bariloche, Quintral 1250, S.C. de Bariloche
(CP: 8400), Rı́o Negro, Argentina.
Tel.: +54 2944 428505/423374 (int 102);
fax: +54 2944 422111;
e-mail: [email protected]
Received 24 May 2011; revised 8 November
2011; accepted 15 December 2011.
Final version published online 3 February
2012.
MICROBIOLOGY ECOLOGY
DOI: 10.1111/j.1574-6941.2011.01287.x
Editor: Philippe Lemanceau
Keywords
Raulı́; phyllosphere; tree domestication;
noncommercial fruits; fruit-borne fungi;
potential biocontrol agents.
Abstract
Nothofagus nervosa (Raulı́) is a native tree species that yields valuable timber. It
was overexploited in the past and is currently included in domestication and
conservation programs. Several research programs have focused on the characterization of epiphytic microorganisms because it has been demonstrated that
they can affect plant–pathogen interactions and/or promote plant growth.
Although the microbial ecology of leaves has been well studied, less is known
about microorganisms occurring on seeds and noncommercial fruits. In this
work, we analyzed the yeast and yeast-like fungi present on N. nervosa fruits
destined for the propagation of this species, as well as the effects of fruit preservation and seed dormancy-breaking processes on fungal diversity. Morphological and molecular methods were used, and differences between fungal
communities were analyzed using a similarity index. A total of 171 isolates corresponding to 17 species were recovered, most of which belong to the phylum
Ascomycota. The majority of the species develop mycelia, produce pigments
and mycosporines, and these adaptation strategies are discussed. It was
observed that the preservation process considerably reduced yeast and yeast-like
fungal diversity. This is the first study concerning microbial communities associated with this ecologically and economically important species, and the information presented is relevant to domestication programs.
Introduction
Microorganisms are capable of synthesizing metabolites
that are of great relevance to industry, such as enzymes,
fatty acids, pigments, and antibiotics. Microbial biodiversity is therefore considered one of the principal sources of
innovation in biotechnology, and a variety of habitats
have been screened to find biotechnologically important
microorganisms (Bull et al., 1992; Middelhoven, 1997;
Bhadra et al., 2008), such as those that could be of
importance for plant improvement and the biocontrol of
plant diseases.
There are approximately 200 000 species of vascular
plants on record, and their surfaces constitute an important habitat for microorganisms, providing a wide range
of microclimatic conditions for correspondingly diverse
microbial communities (Andrews & Harris, 2000; Kowalchuk et al., 2010). The phyllosphere is broadly defined as
FEMS Microbiol Ecol 80 (2012) 179–192
the surfaces and internal parts of aerial plant structures,
including flowers, fruits, stems, and leaves (TimmsWilson et al., 2006; Whipps et al., 2008). It is a harsh
environment with reduced access to nutrients, high fluctuations in temperature and water availability, and exposure to wind and UV radiation (Kowalchuk et al., 2010).
Microorganisms present in the phyllosphere are so
numerous that they can influence plant fitness and contribute to important global processes, such as the carbon
and nitrogen cycles (Andrews & Harris, 2000; Lindow &
Brandl, 2003; Kowalchuk et al., 2010). Consequently, they
may affect the quality and productivity of agricultural
crops (Whipps et al., 2008), either by promoting plant
growth (Glickmann et al., 1998; Brandl et al., 2001),
increasing drought tolerance, and/or playing a major role
in antagonizing pathogens. Hence, the phyllosphere represents a habitat with ecological and biotechnological significance, and a better understanding of this environment
ª 2011 Federation of European Microbiological Societies
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180
may provide new insights into the development of
control strategies for the management of plant diseases
(Lindow & Brandl, 2003; Whipps et al., 2008).
The most abundant inhabitants of the phyllosphere,
and the most studied group, are the bacteria. Yeasts and
yeast-like fungi (These fungi are also known as dimorfic
fungi or black yeasts, both being technical terms to
describe groups of fungi that are quite heterogeneous
from a taxonomic and phylogenetic point of view, most
of them being ascomycetous species. The former corresponds to those fungi that can change from the yeast
form to the mycelial form in response to changes in environmental factors. Black yeasts have melanized cell walls
and most of them exhibit mycelial growth and generate
conidia (Sterflinger, 2006) are also active phyllosphere
colonizers and belong to the largest group of fungi that
grow in this environment, whereas filamentous fungi are
transient inhabitants present mainly as dormant spores
(Andrews & Harris, 2000; Lindow & Brandl, 2003; Whipps et al., 2008). According to Glushakova & Chernov
(2007), plant exudates are the main source of nutrients
for yeasts and yeast-like fungi, and through consumption
of these exudates, they can, in turn, stimulate plant
metabolism. Some studies have also shown that selected
members of the phyllosphere yeast community inhibit or
limit infection of certain plant pathogens (e.g. Fokkema
et al., 1979; Janisiewicz, 1991; Elad et al., 1994; Punja &
Utkhede, 2003; Buck, 2004; Pusey et al., 2009) through
different modes of action, such as niche occupation, competition for nutrients, and antibiosis (Jacobsen, 2006). In
fact, some species such as Aureobasidium pullulans (de
Bary) G. Arnaud, Kloeckera apiculata (Reess) Janke, Pichia
guilliermondii Wick. and Sporobolomyces roseus Kluyver &
C.B. Niel have already been used as biocontrol agents to
manage postharvest losses and control diseases on pear,
citrus fruit, grape, peach, apple, sweet cherry, geranium,
beans and tomato (McLaughlin et al., 1992; Droby et al.,
1993; Elad et al., 1994; Janisiewicz et al., 1994; ChandGoyal & Spotts, 1996a, b; Buck, 2004; Jacobsen, 2006;
Elwakil et al., 2009). Some yeasts have also been used for
the biological control of different diseases associated with
leaves and flowers (Dik & Fokkema, 1993; Urquhart &
Punja, 1997; Tatagiba et al., 1998; Paulitz & Bélanger,
2001; El-Mehalawy, 2004; Pusey et al., 2009).
Many research programs have focused on the characterization of microorganisms that live epiphytically on
leaves, the dominant aerial plant structure (Andrews &
Harris, 2000; Lindow & Brandl, 2003; Jacobsen, 2006;
Sláviková et al., 2007; Pusey et al., 2009). The microbiology of buds and flowers has also been well studied,
mainly because this is the site of infection by several plant
pathogens (Andrews & Harris, 2000; Pusey et al., 2009).
However, little is known about microbial populations on
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N.V. Fernández et al.
seeds and noncommercial fruits (Janisiewicz et al., 2010),
which might be also susceptible to deterioration caused
by insects and different microorganisms, mainly during
maturation and postharvest storage (Marchelli & Gallo,
1999; Hadanich et al., 2008). Most of these microorganisms cause visible symptoms and/or reduce seed germination (Richarson, 1979; McGee, 1995). On the other hand,
some studies have demonstrated that applying microorganisms to seeds may improve plant establishment,
health and growth, particularly if they subsequently
become established in the root zone (Wright et al., 2003;
Bennett & Whipps, 2008).
Several species belonging to the genus Nothofagus constitute the main component of South American temperate forests. Nothofagus nervosa (Phil.) Dim. et Mil.
(Raulı́) is one of the most economically important species of these forests. As it yields a highly valuable wood,
resembling that of Fagus sylvatica L., it was overexploited
in the past and natural populations were drastically
reduced. This critical situation led to the implementation
of conservation and domestication programs. The main
purpose of these programs is to propagate N. nervosa in
nurseries and use them subsequently for reforestation
(Marchelli & Gallo, 1999; Gallo et al., 2004). To accomplish this, seeds are needed, and they are collected
directly from natural populations. All Nothofagus species
have indehiscent dry fruits containing only one seed
(nuts). As the pericarp does not split open, seedlings are
cultivated directly from these fruits, which are not previously disinfected. It is, therefore, expected that the microbiota present on them would be carried through the
cultivation system.
One of the most important diseases affecting Nothofagus propagation in nurseries is the damping-off caused by
different species of Fusarium, Rhizoctonia, Phytophthora,
and Sclerotinia (Azpilicueta et al., 2010), which are common greenhouse pathogens worldwide (Paulitz & Bélanger, 2001; Azpilicueta et al., 2010). These fungi can
seriously reduce seed germination and seedling emergence, stand, and vigor. The use of fungicides is the conventional method for protecting plants against this
disease, but this has resulted in resistance development in
pathogens and most of them are environmentally harmful. Biocontrol methods provide an alternative to chemical fungicides (Berger et al., 1996) and different
microorganisms that are antagonistic against the fungi
that cause damping-off have been found (Berger et al.,
1996; Mao et al., 1997), including some yeast and yeastlike fungi (El-Tarabily, 2004; El-Mehalawy et al., 2007;
Elwakil et al., 2009).
Our main objective was to determine the diversity of
yeast and yeast-like fungi present on N. nervosa fruits
prior to cultivation in nurseries and to evaluate how
FEMS Microbiol Ecol 80 (2012) 179–192
181
Yeast and yeast-like fungi on Nothofagus nervosa fruits
previous manipulation (preservation and process used to
break seed dormancy) influences the occurrence of these
organisms on the fruits. Morphological and molecular
tools were used to characterize these fungi, and differences in fungal diversity between treatments were evaluated. A long-term aim of this work is to identify potential
biocontrol or growth promoting yeasts and yeast-like
fungi that could be of importance for conservation and
domestication programs.
Materials and methods
Fruit harvest
Fruits were collected from N. nervosa trees in a forest situated in the Yuco region (Lacar lake watershed, 40°07′48″
S, 71°34′48″W, Neuquén province, Patagonia, Argentina).
The average age of the fruit-contributing trees was
between 100 and 150 years.
Fruit collection was performed during the fruit-fall season (March–April) in 2002 and 2007 by placing nets
below N. nervosa canopy at approximately 1.5 m above
the ground. Nets were distributed to capture fruits from
a minimum of 40 trees. Fruits were kept in plastic bags
in a cold (2–4 °C), dry place until further procedures
were performed (seeds are still viable after 6–9 years
under these conditions) (Martinez & Schinelli, 2009). It is
important to mention that, in this work, fruit collection
and handling did not differ from the procedures commonly carried out for domestication programs.
fruits of those previously selected were placed in cold
running water for 5 days before isolation.
There were four treatments for yeast and yeast-like
fungi isolation: fresh nonwashed (FnW), fresh washed
(FW), preserved nonwashed (PnW), and preserved
washed (PW) seeds. Each fruit was put into a sterile plastic tube containing 1 mL of sterile 0.9% NaCl solution
and 0.15 g of sterile sand. Tubes were vortex-mixed at
maximum speed for 2 min and centrifuged for 30 s.
Aliquots of 100 lL of the supernatant were surface plated
on MYP medium (malt extract 0.7, yeast extract 0.05,
peptone-soytone 0.25 and agar 1.5% w/w) supplemented
with 0.01% chloramphenicol. Plates were incubated at
room temperature (20 °C) for 72 h and then at 4 °C for
48 h to enhance the development of characteristic colony
color and other morphological features. Three representative colonies of each morphological type present were
picked per plate, and pure isolates were obtained by
repeated streaking on fresh MYP medium. All the isolates
were first grouped according to their macromorphology
(color, colony shape and texture, and presence of mycelia) and their ability to produce mycosporines. Mycosporine light induction, extraction, and analysis were assessed
in collaboration with Dr. Martı́n Moliné (Laboratorio de
Microbiologı́a Aplicada y Biotecnologı́a, Centro Regional
Universitario Bariloche) and according to Moliné et al.
(2011). Isolates were cryopreserved in 20% glycerol and
stored at 80 °C.
Molecular analyses
Yeasts isolation and characterization
DNA extraction and PCR fingerprinting
To evaluate whether preservation at 4 °C influences yeast
and yeast-like fungal diversity, both fresh and preserved
fruits were analyzed. Fresh fruits were collected some
weeks before this work was performed (harvest 2007),
while the preserved fruits were part of a harvest that had
been stored at 4 °C for 5 years (harvest 2002). For yeast
and yeast-like fungi isolation, 30 fresh and 30 preserved
fruits were randomly selected from each pool.
For seed germination, it is first necessary to break seed
dormancy by placing the fruits in cold running water
(c. 5 °C) for 5 days (washing process). Another important requirement is that the seeds have to be sowed no
more than 1 cm below the surface, in a porous substrate
that allows hydration and avoids compaction. The optimum temperature for seed germination is 18–21 °C. In
these conditions, seedlings emerge in 10–15 days (Schinelli & Martinez, 2010). To evaluate whether microbial
composition changes after the washing process and to
describe the yeast and yeast-like fungi present on the
fruits at the time of sowing, 15 fresh and 15 preserved
For total genomic DNA extraction, a loopful of a fresh
culture of each isolate (cultivated in MYP – malt–yeast–
peptone agar – at 20 °C for 72 h) was transferred to
100 lL of sterile pure water, frozen at 80 °C for an
hour, and heated at 100 °C for 10 min. Tubes were centrifuged for 1 min at 13 200 g, and the supernatant was
transferred to a clean tube.
All the isolates within the different morphological
groups were subjected to PCR fingerprinting, employing
the mini/microsatellite-primed PCR technique (MSP-PCR)
and the M13 primer (5′-GAGGGTGGCGGTTCT-3′,
Sigma). PCR reaction was performed in a total volume of
25 lL containing 3 mM MgCl2, 0.2 mM dNTPs, 0.8 lM
primer, 1 U Taq polymerase (Invitrogen), 19 of the reaction buffer, and 5 lL of a 1 : 175 genomic DNA dilution
(Libkind et al., 2003; de Garcı́a et al., 2007). The amplification was carried out in a Multigene Labnet cycler according
to the following PCR conditions: an initial denaturing step
at 95 °C for 5 min, followed by 40 cycles of 45 s at 93 °C,
60 s at 55 °C and 60 s at 72 °C, and a final extension step
FEMS Microbiol Ecol 80 (2012) 179–192
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
182
of 6 min at 72 °C. Amplified DNA fragments were separated by electrophoresis in 1.5% (w/v) agarose gels.
Sequencing and phylogenetic analysis
Isolates with identical DNA banding patterns were
grouped together, and at least two representatives of each
group were sequenced. For DNA sequencing, the D1/D2
domain of the 26S rRNA gene was amplified using the
NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and
NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) primers (Genbiotech, Argentina). PCR was performed with 0.5 mM
MgCl2, 0.8 mM dNTPs, 0.2 lM each primer, 1 U Taq
polymerase, and 19 reaction buffer (Invitrogen), and
3.8 lL of the 1:500 genomic DNA dilution was added to
the reaction. PCR conditions were as follows: a denaturing step at 95 °C for 2 min, 35 cycles of 15 s at 95 °C,
25 s at 54 °C and 20 s at 72 °C, and a final extension
step of 10 min at 72 °C. Sequencing was performed from
purified PCR products (Wisard kit; Promega) using BigDye chemistry (Applied Biosystems) and analyzed on
ABI3130XL automatic sequencers (Genomic Unit, CNIA
INTA Castelar facilities, Argentina).
Sequences were manually corrected, aligned, and subjected to phylogenetic analysis using the Molecular Evolutionary Genetics Analysis software (MEGA4). Two
phylogenetic trees, one for Ascomycetes and the other for
Basidiomycetes, were constructed using the neighbor-joining algorithm. Bootstrap values were calculated from
1000 replicates and the Kimura 2-parameter model to
estimate evolutionary distance. If several strains of a single species were sequenced, only four were taken into
account for constructing the tree (if there were sequences
belonging to the same species but with some nucleotidic
differences – 5 or less – those with the higher number of
differences were selected). On the phylogenetic tree, all
the species were clustered with their nearest phylogenetic
relatives according to the NCBI database (http://www.
ncbi.nlm.nih.gov) and with type strains if they were available. All the nucleotide sequences obtained in this work
were deposited in the NCBI GenBank database and are
available for comparative purposes under the accession
numbers HQ629551–HQ629619 (Table S1). The Index
Fungorum database was used for nomenclature and classification of the species (www.indexfungorum.org).
Community analysis
Beta-diversity is defined as the variation of species composition over space and time (Anderson et al., 2006) and is
crucial to the understanding of how environmental factors
affect biodiversity, even in microbial populations. The
modified Jaccard index described by Chao et al. (2005)
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Published by Blackwell Publishing Ltd. All rights reserved
N.V. Fernández et al.
takes species abundance into account, being better suited
than the corresponding classic index for the assessment of
compositional similarity between samples containing
numerous rare species (Chao et al., 2005), which is the
case in this work. This index was selected for measuring
beta-diversity and for evaluating how the preservation and
washing processes affected yeast and yeast-like fungal
composition associated with N. nervosa fruits. Differences
in yeast and yeast-like fungal communities were analyzed
between: (a) fresh and preserved fruits, (b) FnW and FW
fruits, and (c) PnW and PW fruits.
Statistical analysis
A chi-squared analysis was carried out to test the association between the categorical variables of preservation
(fresh and preserved) and washing process (nonwashed
and washed). To evaluate whether these treatments modify the yeast and yeast-like fungi abundance on the fruits,
two Paired t-tests were also conducted (fresh vs. conserved and nonwashed vs. washed).
Results
A total of 171 isolates were recovered from the 60 N.
nervosa fruits examined: 94% from fresh and 6% from
preserved fruits (Table 1). Yeast and yeast-like fungi were
present in all the fresh but only in 20% of the preserved
fruits. The percentage of isolates recovered from nonwashed and washed treatments were 47% and 53%,
respectively. The chi-squared test showed that there was
no association between these variables (preservation and
washing) (v2 = 0.69, P < 0.05), and the Paired t-test indicated that the preservation process significantly reduced
the quantity of yeast and yeast-like fungi present on the
fruits analyzed (P = 0.013), whereas the washing treatment did not bring about a change (P = 0.170).
Seventeen yeasts and yeast-like fungi species were identified using a four-step strategy that involved: (i) allocation of isolates into distinct morphological groups,
(ii) genomic profiling within each morphological group
using MSP-PCR fingerprinting (M13 primer), (iii)
sequencing of the D1/D2 domain of 26S rRNA gene for
representatives of each genomic group, and (iv) species
allocation of isolates by phylogenetic positioning, using
sequences of reference strains available in databases. Yeasts
belonging to the phylum Ascomycota were dominant, both
at an isolate (78%) and a species level (61%), being five of
them new Ascomycetous species. The remaining species
belong to the phylum Basidiomycota (Table 1).
All the Ascomycetous species isolated in this work are
dark pigmented (green, brown, and black), melanincontaining yeast-like fungi capable of forming conspicuous
FEMS Microbiol Ecol 80 (2012) 179–192
183
Yeast and yeast-like fungi on Nothofagus nervosa fruits
Table 1. Taxa and number of strains isolated per species in each treatment
Fresh
Taxa
Ascomycota
Dothideales
Aureobasidium pullulans (de Bary) G. Arnaud
Dothichiza sp. 1
Dothichiza sp. 2
Coniochaetales
Lecythophora mutabilis (J.F.H. Beyma) W. Gams & McGinnis
Taphrinales
Taphrina wiesneri (Ráthay) Mix
Chaetothyriales
Phaeomoniella zymoides Hyang B. Lee, J.Y. Park, Summerb. & H.S. Jung
Phaeomoniella sp.
Incertae sedis
Ascomycetous yeast sp. 1
Ascomycetous yeast sp. 2
Coniozyma leucospermi (Crous & Denman) Crous
Basidiomycota
Tremellales
Cryptococcus adeliensis Scorzetti, I. Petrescu, Yarrow & Fell
Cryptococcus diffluens (Zach) Lodder & Kreger-van Rij
Cryptococcus wieringae Á. Fonseca, Scorzetti & Fell
Cryptococcus heveanensis (Groen.) Baptist & Kurtzman
Trichosporon dulcitum (Berkhout) Weijman
Sporidiobolales
Rhodotorula colostri (T. Castelli) Lodder
Rhodotorula fujisanensis (Soneda) E.A. Johnson & Phaff
Strains isolated per treatment
Preserved
nW
W
nW
W
15
26
1
14
21
–
1
–
–
1
–
–
1
–
2
–
1
1
1
Pig
Myc
Mcl
31 (18%)
47 (27%)
1 (< 1%)
+M
+M
+M
+
+
+
+
+
+
–
3 (2%)
+M
–
+
–
–
1 (< 1%)
+M
+
+
–
–
–
–
–
–
1 (< 1%)
1 (< 1%)
+M
+M
+
+
+
+
19
–
–
27
–
1
–
–
–
–
1
–
46 (27%)
1 (< 1%)
1 (< 1%)
+M
+M
+M
+
–
+
+
+
+
–
1
1
11
–
2
–
–
8
1
–
–
–
–
–
–
–
–
1
–
2
1
1
20
1
–
–
–
–
–
–
+
+
+
+
–
–
–
–
+
–
–
77
161
1
8
84
–
–
3
10
3
1
7
+C
–
–
–
–
–
W, washed; nW, nonwashed; %, percentage of the total number of isolates; Pig, pigments;
mycelia.
mycelium. Most of the basidiomycetous isolates are nonpigmented and lack mycelia, with the exception of Rhodotorula colostri that is a pink carotenogenic yeast, and
Trichosporon dulcitum, which develops mycelia. Seventyone percent of the total number of species produces
mycosporines, including the most abundant ones (Table 1).
Dothichiza sp. 1, Ascomycetous yeast sp. 1, A. pullulans
(Ascomycota) and Cryptococcus heveanensis (Basidiomycota) were the most abundant species (Table 1).
Altogether, these four species accounted for 84% of the
total number of isolates. Aureobasidium pullulans was the
only species present in all the treatments, and C. heveanensis was found in three of them. Both species were present more frequently on fresh fruits. Dothichiza sp. 1 and
Ascomycetous yeast sp. 1 were recovered only from fresh
fruits. Lecythophora mutabilis was present only on nonwashed fruits, while R. colostri and Rhodotorula fujisanensis appeared exclusively on washed fruits (Table 1). The
remaining species (59%), were represented by no more
than one or two isolates and were found in only one
treatment, mostly corresponding to fresh fruits.
FEMS Microbiol Ecol 80 (2012) 179–192
M
Total (%)
(1%)
(< 1%)
(< 1%)
(12%)
(< 1%)
4 (2%)
9 (5%)
171
C
, melanin; , carotenes; Myc, mycosporines; Mcl,
The different clusters on the phylogenetic trees correspond to the orders to which the yeasts and yeast-like
fungi analyzed in this study belong (Fig. 1). Figure 1a
shows that the yeast-like fungi Ascomycetous yeast sp. 2 is
close to the L. mutabilis cluster (Fig. 1a), but there are 10
nucleotidic differences distinguishing it from this species,
so that it represents a new species. Both species lack the
ability to produce mycosporines (Table 1). Ascomycetous
yeast sp. 1 is also a new species, and there are eight nucleotidic differences between it and its closest relative
(Fungal Endophyte 9096), which is also an undescribed
species. Aureobasidium pullulans strains are situated in
different subclusters on the same branch, suggesting that
there are different varieties of this species among the
strains recovered in this work. Dothichiza sp. 1 and Dothichiza sp. 2 are separated from Dothichiza pithyophila by
13 and 17 nucleotidic differences, respectively, thus clearly
phylogenetically separated from this species and from
each other. In the tree corresponding to the phylum Ascomycota, it can also be observed that Phaeomoniella sp. 1
is separated from the Phaeomoniella zymoides cluster,
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184
N.V. Fernández et al.
(a)
(b)
Fig. 1. Phylogenetic tree obtained by neighbor-joining and the Kimura 2-parameter analysis of the D1/D2 domains of the 26S rRNA gene. The
numbers on the branches are the frequencies with which a given branch appeared in 1000 bootstrap replications (values smaller than 50% are
not shown). Sequences determined in this study are in bold. Additional sequences were retrieved from the GenBank. Fonsecaea compacta and
Tilletiaria anomala were used as outgroups for the yeasts and yeast-like fungi present on Nothofagus nervosa fruits, belonging to the (a) phylum
Ascomycota and (b) phylum Basidiomycota, respectively. The following symbols indicate the substrate from which each strain was isolated:
= plant associated,
= soil associated,
= aquatic environments,
= others, T = Type strain.
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FEMS Microbiol Ecol 80 (2012) 179–192
185
Yeast and yeast-like fungi on Nothofagus nervosa fruits
which suggests that it is a different species (Fig. 1a). The
phylogenetic tree corresponding to the phylum Basidiomycota (Fig. 1b) shows that C. heveanensis is closer to
T. dulcitum than to the other Cryptoccocus. Rhodotorula
colostri and R. fujisanensis are included in the same order
and do not produce mycosporines (Table 1). It can be
observed in these trees that most of the nearest phylogenetic neighbors were also isolated from plants or soil.
The modified Jaccard index showed low values when the
comparisons between fresh/preserved and PnW/PW fruits
were made (0.29 and 0.11, respectively), indicating that the
yeast communities found on the fruits were very different
following these treatments. In contrast, this abundancebased index indicated that fungal communities associated
with FnW and FW fruits were similar (78%) (Table 2).
Discussion
Yeast and yeast-like fungus diversity
The method used in this work for assessing yeast and
yeast-like fungus diversity on N. nervosa fruits consisted
of: isolating these fungi according to standard techniques
and sorting all the isolates into groups based on macroscopic characteristics and mycosporine production; each
group was further characterized using the MSP-PCR fingerprinting method and the M13 primer, which usually
generates species-specific patterns; finally, representative
strains of the different MSP-PCR groups were selected for
DNA sequencing. This strategy has been used in other
studies on genetic diversity among species and genera
(Balefires Couto et al., 1996; Meyer et al., 2001; Gadanho
& Sampaio, 2002; Libkind, 2007) and also as a tool for
yeast diversity description in natural environments (Gadanho et al., 2003; Libkind et al., 2003; de Garcı́a et al.,
2007; Muñoz, 2010; Mestre et al., 2011). One of the main
reasons this strategy is suitable for this type of study, and
Table 2. Community analysis of the yeast and yeast-like fungi
present on fruits before and after the preservation and washing
processes
Beta indices
No. shared
species
No. unique
species
Shared species
frequencies
Modified
Jaccard index
Fresh
(16)–Preserved (6)
Fresh
nW(10)–W(10)
Preserved
nW(2)–W(5)
5
4
1
11–1
6–6
1–4
0.35–0.60
0.92–0.83
0.33–0.14
0.29
0.78
0.11
nW, nonwashed fruits; W, washed fruits; (no.), number of species per
treatment.
FEMS Microbiol Ecol 80 (2012) 179–192
so widely used, is that it allows rapid and inexpensive
genetic characterization of several isolates at the same
time (Libkind, 2007).
Some authors observed that Ascomycetous and basidiomycetous yeasts and yeast-like fungi were present in
approximately equal frequencies on leaves (Sláviková
et al., 2007) and bark (Bhadra et al., 2008) of different
tree species. However, Fonseca & Inácio (2006) suggested
that one of the most significant trends to emerge from
the analysis of several studies carried out in the phyllosphere is the clear dominance of Basidiomycetes. These
authors argue that the microenvironments present on aerial plant organs are analogous (leaves, stems, most fruits,
and flowers), so it is not surprising that the dynamics
and composition of the respective yeast communities are
also similar. Nevertheless, in this work, 17 species of
yeasts and yeast-like fungi were isolated from N. nervosa
fruits, the Ascomycetes being dominant both at isolate
(78%) and species level (61%). These fruits are not fleshy
and do not have stomata or water retaining structures, so
they have lower humidity conditions than other plant
organs. These results are in agreement with Middelhoven
(1997) and Bhadra et al. (2008), who also found that
Ascomycetous yeast and yeast-like fungi were dominant in
the phyllosphere of 24 plant species in an arid climate
and in different species of tree bark from a forest in
India. Taking this information into account, plus the fact
that Ascomycetes seem to be more stress resistant than
Basidiomycetes (Baar et al., 1999; Gorbushina & Broughton, 2009), it can be suggested that Ascomycetous yeast
and yeast-like fungi are better adapted to low water availability and desiccation than the basidiomycetous (Bhadra
et al., 2008), thus explaining the higher incidence of ascomycetes in the microbiota of N. nervosa indehiscent dry
fruits.
Most of the species present on N. nervosa fruits have
been previously found on plants (Fig. 1; Weber et al.,
2002; Glushakova & Chernov, 2004; Muñoz, 2010), except
for L. mutabilis and T. dulcitum, which have been isolated
from soil. Most of the T. dulcitum strains found in the
CBS database and in other studies (Mestre et al., 2011)
have also been isolated from soil, suggesting that this is
the primary habitat of this species and that it reaches aerial plant parts by vectors (small mammals, birds, and
insects) and/or wind. Bhadra et al. (2008) also found that
most of the nearest phylogenetic neighbors to the yeast
groups isolated from tree barks were isolated in association with plants and their environment (fruits, fruit
juices, plant extracts, insects, and soil). The occurrence of
soil yeasts in the phyllosphere is not rare, according to
Andrews & Harris (2000), who stated that species found
in the rhizosphere are commonly present in the phylloplane.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
186
Aureobasidium pullulans was the only species present
in all the treatments. This is a cosmopolitan yeast-like
fungus that has been isolated from diverse substrates
and seems to be ubiquitous on aerial plant surfaces
worldwide (Fonseca & Inácio, 2006; Janisiewicz et al.,
2010; Muñoz, 2010). Some species found in this study
have been previously recorded in other substrates from
Patagonia. Rhodotorula colostri, R. fujisanensis, and T.
dulcitum have also been reported from Nothofagus forest
soil (Mestre et al., 2011), while R. colostri and Cryptococcus adeliensis have been found in aquatic environments
of glacial origin in northern Andean Patagonia (de Garcı́a et al., 2007).
Only four of the species identified in this work (24%)
were dominant at community level. This observation is in
accordance with different authors who stated that populations in the phyllosphere are commonly dominated by a
few species. Rare species account for a significant proportion of the species richness present in this environment,
and many novel taxa are present among them (Lindow &
Brandl, 2003; Glushakova & Chernov, 2004; Fonseca &
Inácio, 2006; Bhadra et al., 2008; Whipps et al., 2008;
Janisiewicz et al., 2010). Five of the species isolated from
N. nervosa fruits (29%) have not been previously
described; two of which are the most abundant species
(Dothichiza sp. 1 and Ascomycetous yeast sp. 1). This
information suggests that different plant species harbor
highly adapted microorganisms, which might have novel
biotechnological capacities (Kowalchuk et al., 2010).
Effects of the preservation and washing
processes
Following the arrival of microbial cells in the phyllosphere, a variety of factors determine whether they are
able to colonize and survive on the plant surface, mostly
substrate characteristics and environmental conditions
(Whipps et al., 2008). For example, the growth of microorganisms in this environment is supported by nutrients
leaking from the plant, as well as external sources, like
pollen deposits, organic debris, and honeydew (Glushakova & Chernov, 2007; Whipps et al., 2008). If the supply
of nutrients is insufficient, most of the microorganisms
present on the plant organ will not be able to survive for a
long period of time. Temperature and water availability
are also important factors that regulate microbial diversity.
Some authors observed that fungal diversity decreases during the winter and reaches a minimum at the beginning of
spring, probably as a consequence of microbial intolerance
to low temperatures and desiccation, and/or to a decrease
in airborne inoculum and nutrients (Buck et al., 1998;
Glushakova & Chernov, 2007). When N. nervosa fruits are
preserved, microorganisms living on them are exposed to
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
N.V. Fernández et al.
low temperatures for long periods of time, without nutrient, water, or inoculum input. Under these conditions, it
is to be expected that preservation will influence the abundance and composition of fruit-borne yeast and yeast-like
fungi, as observed in this study, where significantly lower
numbers of isolates and species were registered on preserved than on fresh fruits (Table 2).
A conspicuous feature of aerial plant surfaces is that
free moisture is quite transient, mainly associated with
rain and dew. Consequently, water availability significantly influences microbial communities in the phyllosphere. For instance, when the fern Polypodium
polypodioides (L.) Watt is exposed to rainfall after a period of desiccation, the complex phyllosphere community
undergoes changes in overall structure and activity (Jackson et al., 2006). In N. nervosa, we observed that yeast
and yeast-like fungal communities tended to differ
between PnW and PW fruits (only two species were isolated before the washing treatment and five after it, with
only one in common). When FnW and FW fruits are
compared, we see that species composition also differs
(six species disappeared during the washing process but
another six emerged), although communities are similar
because the most abundant species are the same for both
treatments (A. pullulans, Dothichiza sp. 1, Ascomycetous
yeast sp. 1, and C. heveanensis) (Tables 1 and 2). One
possible explanation for this phenomenon is that species
better adapted to high moisture conditions or those that
need high water availability emerge (e.g. R. colostri and
R. fujisanensis that were recovered from fresh and preserved fruits only after the washing treatment), while
those not tolerant to this condition do not survive (e.g.
L. mutabilis that was present in both types of fruits before
the washing treatment, but not after it). It is also possible
that the washing treatment removed some competitive
species, so others were able to emerge. These findings
suggest that the washing process alter the yeast and yeastlike fungal composition present on the fruits, but not the
overall community structure.
These results indicate that customary procedures during N. nervosa propagation (such as storage at 4 °C and
the washing process for breaking seed dormancy) alter
microbial communities present on the fruit, and this
information is relevant to domestication programs. If
native microbiota present on fresh fruits benefits
germination or seedling stand, then fruits should not be
preserved at 4 °C for long periods of time.
Adaptations to the phyllosphere environment
Resident phyllosphere microorganisms are presumably
endowed with suitable niche-specific traits for survival
and growth on their particular surface habitats. Some
FEMS Microbiol Ecol 80 (2012) 179–192
Yeast and yeast-like fungi on Nothofagus nervosa fruits
of these attributes include high growth rates, the ability
to compete for nutrients and to withstand periods
of drought, and varying osmotic and temperature
conditions. Some of the specific adaptive properties that
epiphytic yeasts possess are a capacity for substrate adherence (mycelia, capsules) and pigmentation (carotenes,
melanin) (Andrews & Harris, 2000; Lindow & Brandl,
2003; Kowalchuk et al., 2010).
It has been demonstrated in Candida spp. that adherence is largely influenced by mycelium development, associated with high attachment capacity for tissue
colonization (Trochin et al., 1991). Most of the species
described in this study, except for those included in the
Cryptococcus and Rhodotorula genera, are capable of forming conspicuous mycelia, suggesting that this structure is a
widespread attachment strategy among the yeast and
yeast-like fungi present on N. nervosa fruits. Another common trait described for phylloplane yeasts is the production of capsules, mainly in Cryptococcus and Rhodotorula
species, which lack mycelium (Golubev, 1991; Glushakova
& Chernov, 2004). Experimental evidence suggests that
this structure increases yeast fitness and survival when
subject to stress and protects them from drying out during low water activity (Golubev, 1991; Bhadra et al., 2008).
Capsules also play an important adhesion role (Deak,
2006) and seem to explain, at least in part, the observed
differences between resident and transient microorganisms
in the phyllosphere (Fonseca & Inácio, 2006). The species
described in this work, then, have various adhesion
strategies that allow them to survive in this extreme environment, exposed to rain and wind, and remain attached
to the substrate even after the washing treatment.
Most phyllosphere microorganisms are capable of withstanding high UV light levels, mainly because of two mechanisms: pigmentation and DNA repair (Kowalchuk et al.,
2010). Yeasts can be damaged by UV wavelengths of sunlight, so pigmented species are abundant on plant surfaces,
mostly corresponding to the genera Rhodothorula and
Sporobolomyces (Fonseca & Inácio, 2006). These organisms
contain red, orange, and pink carotenoid pigments, which
provide indirect protection for the cells by quenching the
reactive oxygen species produced by radiation (Young,
1991). In this work, R. colostri was the only species belonging to this group. However, a high proportion of species
(53%) were found to be darkly pigmented as a consequence
of their melanin content (Table 1). Melanins are not only
responsible for the dark-green, brown, and black color of
the fungi but also for a number of properties helping them
to survive under conditions of environmental stress, such
as temperature and osmotic extremes, UV radiation, and
desiccation (Sterflinger, 2006).
Mycosporines are hydrosoluble molecules with UV
absorption at wavelengths mainly around 310 nm (BandarFEMS Microbiol Ecol 80 (2012) 179–192
187
anayake, 1998). Several yeast species are able to synthesize
and accumulate UV-radiation-absorbing mycosporine
metabolites, such as the mycosporine–glutaminol–glucoside (MGG). This molecule has been shown to play an
important role as a UVB photoprotective metabolite in
yeasts by protecting them against direct DNA damage
(Moliné et al., 2011). This idea is supported by the fact that
most of the species isolated from N. nervosa fruits (71%),
which in nature are generally exposed to high levels of solar
radiation, synthesize mycosporines. Another interesting
observation is that mycosporinogenesis seems to be a feature related to certain phylogenetic groups (taxon-specific),
as was suggested by Libkind et al. (2005). Our findings are
in agreement with these authors, because species included
in the orders Dothideales and Chaetothyriales constitutively
synthesize mycosporines, while those belonging to the
orders Coniochaetales and Sporidiobolales are not able to do
so. The order Tremellales is highly heterogeneous and polyphyletic, so it is not surprising that the ability to produce
mycosporines varied among the isolated species included
in it (Fig. 1).
As the phyllosphere environment is not homogenous,
resident microbial populations present a marked variation
in exposure to light, wind, rainfall, and airborne inoculum (Deak, 2006; Fonseca & Inácio, 2006). Although
many aerial structures of trees are usually within or
beneath the tree canopy, most of the yeasts present in
N. nervosa fruits are darkly pigmented and mycosporineproducing species. Consequently, it would be highly
probable that these molecules (carotenes, melanin, and
mycosporines) have another biological function in addition to UV protection. Nothofagus fruits are usually
exposed to prolonged desiccation periods, and it is
known that desiccation is an important factor that causes
oxidative stress (Alpert, 2006). Carotenes, melanin, and
mycosporines are known to have antioxidant properties,
so it is possible that these molecules play an antioxidant
role in these microorganisms. This hypothesis is supported by the work carried out by Moliné et al. (2011),
who have recently demonstrated the ability of the
mycosporine-derived glucosides to scavenge or quench
reactive oxygen species, indicating that in these fungi,
mycosporines might play a role in fighting oxidative
stress, in addition to UV protection.
All these findings are in agreement with Muñoz (2010),
who found dark pigmented yeast and yeast-like fungi in
the phylloplane of Nothofagus pumilio, most of which
were also capable of forming mycelia and producing
mycosporines. According to this information, it seems
that in spite of having different community composition,
the yeast and yeast-like fungi present on different substrates within Nothofagus phyllospheres have the same
adaptations to this environment.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
188
Potential biotechnological application of
epiphytic yeast and yeast-like fungi
A basic understanding of the organisms, relationships,
and driving forces that have evolved in the phyllosphere
is crucial to the manipulation of plant-associated microbiota for the development of biocontrol methods that can
contribute to more effective and less environmentally
damaging methods of plant protection (Droby et al.,
1993; Berger et al., 1996; Schoeman et al., 1999; Lindow
& Brandl, 2003). In addition, microorganisms present in
this environment are important sources of diverse compounds with biotechnological applications (Bull et al.,
1992), such as xylanases, cellulases, nitrogenases, lipases,
amylases, pectinases, esterases, proteases (Middelhoven,
1997; de Garcı́a et al., 2007; Bhadra et al., 2008), and
antifungal compounds, such as pyrrolnitrin. Pyrrolnitrin
is produced by different bacteria, including some Burkholderia cepacia isolated from apple leaves (Janisiewicz &
Roitman, 1988; Janisiewicz & Yourman, 1991). This
bacterial metabolite is currently used in some successful
fungicides (e.g. fenpiclonil – Nevill et al., 1988 and
fludioxonil – Gehmann et al., 1990), against fruit decays
produced by different pathogens, such as Fusarium graminearum Schwabe, Botrytis cinerea Pers., Rhizoctonia solani
J.G. Kuhn, and Penicillium expansum Link.
The biological control of plant diseases has been
focused primarily on the use of bacteria or filamentous
fungi (Schoeman et al., 1999). The application of yeast
and yeast-like fungi for controlling plant diseases seems to
be a new trend in this area (El-Sayed & El-Nady, 2008),
despite its potential as biocontrol agents was described a
long time ago (Janisiewics, 1987). These fungi have been
said to provide a natural buffer against infection by several
pathogens (Fokkema et al., 1979; Punja & Utkhede, 2003;
El-Tarabily, 2004), and some attributes that make them
suitable as biocontrol agents are as follows: rapid colonization of surfaces and survival for long periods under varying conditions; the production of extracellular
polysaccharides that enhance their chances of survival and
restrict both colonization sites and the flow of germination cues to pathogen propagules; the use of available
nutrients for rapid proliferation; and minimal reaction to
pesticides (Janisiewicz, 1991). These fungi have shown
great potential for reducing foliar diseases, especially those
caused by mildew fungi (Urquhart & Punja, 1997; El-Mehalawy, 2004), and for effectively inhibiting the development of postharvest pathogens on various fruits
(McLaughlin et al., 1992; Droby et al., 1993; Elad et al.,
1994; Janisiewicz et al., 1994; Chand-Goyal & Spotts,
1996a, b). Among the yeasts that have been described to
have antagonistic effects on plant pathogens are different
Candida, Cryptococcus, Kloeckera, Pichia, Rhodotorula,
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
N.V. Fernández et al.
Sporobolomyces, and Trichosporon species (McLaughlin
et al., 1992; Droby et al., 1993; Chand-Goyal & Spotts,
1996a, b; Buck, 2004; El-Mehalawy, 2004; Medina et al.,
2009; Pusey et al., 2009; Janisiewicz et al., 2010). Moreover, commercial yeast-based formulations have been
developed (see Reglinski et al., 2011), and some are available commercially, such as Bionext (Candida oleophila
Montrocher – Bionext, Belgium and Leasaffe International, France), Shemer (Metschnikowia fructicola Kurtzman & Droby – AgroGreen Israel) and Candifrut
(Candida sake (Saito & M. Ota) Uden & H.R. Buckley ex
S.A. Mey. & Ahearn – IRTA, Spain). Special attention has
to be paid to A. pullulans. This is a ubiquitous yeast-like
fungus present on plant surfaces and several other substrates worldwide, and it has been shown to be antagonistic to different plant pathogens (Schena et al., 2002;
Janisiewicz et al., 2010; Reglinski et al., 2011). This fungus
has been used for the formulation of ecologically harmless
products to control postharvest diseases, such as Boniprotect and Blossom-protect (Bio-protect, Germany).
Greenhouse or nursery conditions (temperature, light,
and fertilizer regimes) are optimized for maximal plant
growth, but they are also favorable for diseases. Pathogens
enter this system via air, irrigation water, insects, contaminated shoes, tools, or equipment. Many pathogens are
also introduced on seeds. Disinfested soil or soilless substrates (peat or rockwool) lack the microbial diversity
responsible for the natural buffering against pathogens, so
soilborne pathogens such as Pythium and Rhizoctonia can
grow and seriously affect seeds and seedlings (Paulitz &
Bélanger, 2001). The success of biocontrol depends on
how the microbial searching and screening process is carried out. For example, finding microorganisms to protect
postharvest fruit is likely to require screening for microorganisms that colonize the surface of the fruit, because a
biocontrol agent must occupy an ecological niche similar
to that of the plant pathogen (Janisiewics, 1987; Paulitz &
Bélanger, 2001; Janisiewicz & Korsten, 2002; Fravel,
2005). In the same way, the screening of yeast and yeastlike fungi present on N. nervosa fruits to be used in nurseries for seedling propagation would be a good strategy
for finding microorganisms with an antagonistic effect
against different phytopathogens, including those causing
damping-off (Mao et al., 1997; El-Mehalawy et al., 2007;
Elwakil et al., 2009). Some of the yeast and yeast-like
fungi isolated in this work belong to genera (Cryptococcus,
Rhodotorula, Trichosporon) or even species (A. pullulans,
R. colostri) that have been successfully used as biocontrol
or growth promoting agents. Thus, future studies will
focus on determining the effect of these microorganisms
on the control of plant pathogens and on improving germination and seedling stand of N. nervosa in domestication programs.
FEMS Microbiol Ecol 80 (2012) 179–192
Yeast and yeast-like fungi on Nothofagus nervosa fruits
Conclusion
This study describes the occurrence of yeasts and yeastlike fungi present on the indehiscent dry fruits of an
ecologically and economically important forestry species.
It contributes to the description of microbial populations
present in N. nervosa phyllosphere as well as to the general knowledge of fungal biodiversity in Patagonia.
The species described in this work have adaptation
strategies that allow them to survive in extreme environments where they are exposed to rain, wind, and high
levels of UV radiation. Most of them are capable of forming mycelia, which allow them to remain attached to the
substrate even after the washing treatment. In addition,
the majority of the species analyzed are pigmented and
can produce mycosporines, which is not surprising
because a high proportion of melanin and carotene-producing microorganisms are associated with environmentally stressed areas, such as hot and cold deserts, alpine
regions, and the upper biosphere (Sterflinger, 2006).
This investigation has revealed that both preservation
and washing processes alter microbial communities on
N. nervosa fruits. The former considerably reduces the
number of yeast and yeast-like fungi, while the washing
process tends to change species composition. The valuable
information obtained through this study constitutes the
first step toward exploring native yeast and yeast-like
fungi for improving seed germination and seedling stand
in N. nervosa domestication programs. More research is
needed, however, to determine the effects of this type of
practice on seed germination, plant improvement, and
biocontrol strategy development.
Acknowledgements
Seed collection within Lanı́n National Park was carried
out by the Ecological Genetics and Forest Tree Breeding
Group with the permission and collaboration of National
Park Administration. We thank BSc. (Hons) Audrey
Urquhart and Lic. Silvia Brizzio for language revision and
Dr Martı́n Moliné for assisting us with mycosporine
induction, extraction, and analysis and for his valuable
comments on this manuscript. This work was partially
financed by the project PNFOR044321 INTA (Domesticación de especies forestales nativas patagónicas) and by
grants B143 (Universidad Nacional del Comahue) and
PICT 22200 (ANPCyT).
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Accession numbers, isolation substrates and
type of fungi corresponding to the nucleotide sequences
obtained in this work (HQ629551–HQ629619) and to the
reference strains. Sequences used for constructing the
trees presented in Figure 1 are indicated in bold.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
FEMS Microbiol Ecol 80 (2012) 179–192